Sun Renjie, Liu Hanze, Sun Siqi, Wang Yating, Shan Ying, Li Xiaoliang, Fang Weihuan, Yang Yongle, Xie Ronghui, Zhao Lingyan
Zhejiang Provincial Center for Animal Disease Control and Prevention, Hangzhou, 311199, China.
China Animal Health and Epidemiology Center, Qingdao, 266032, China.
Virol J. 2025 Feb 1;22(1):23. doi: 10.1186/s12985-025-02625-w.
Porcine circoviruses 3 (PCV3) and 4 (PCV4) are emerging pathogens with global implications for swine industry, disturbing the diagnosis of PCVs associated diseases due to a range of similar clinical symptoms and increasingly coinfections. A rapid and accurate method for detection of PCV3 and PCV4 is critical for controlling the transmission of associated disease.
We developed a duplex real-time recombinase aided amplification (RAA) assay for detection of both PCV3 and PCV4 simultaneously. The assay was completed within 20 min at 39℃ with the designed optimal primers and probes.
The established assay was more convenient and simpler operation compared with conventional molecular biological assays. The assay achieved a detection limit of 73.67 copies/reaction for each circovirus (at 95% probability by probit regression analysis) and showed high specificity and no cross-reactivity with other important porcine viruses (including PCV2). The intra- and inter-group coefficients of variation (CV) were ranged from 2.08 to 4.97%, indicating high stability and reliability. Comparative analysis with PCV3 and PCV4 qPCR on 60 clinical samples and artificially spiked samples indicated high congruence (the kappa value was 0.966 and 1, respectively, with p < 0.001), with only minor discrepancies, validating effectiveness of the duplex RAA assay in detecting co-infections and its suitability for preliminary clinical diagnosis of PCV3 and PCV4.
This study provides a robust basis for multiplex detection of veterinary pathogens using RAA technique, enhancing the field's capacity to control PCV3 and PCV4, and supporting reliable aid for epidemiological understanding of emerging circoviruses.
猪圆环病毒3型（PCV3）和4型（PCV4）是对全球养猪业有影响的新出现病原体，由于一系列相似的临床症状以及越来越多的混合感染，干扰了与猪圆环病毒相关疾病的诊断。一种快速准确检测PCV3和PCV4的方法对于控制相关疾病的传播至关重要。
我们开发了一种双重实时重组酶辅助扩增（RAA）检测方法，用于同时检测PCV3和PCV4。使用设计的最佳引物和探针，该检测在39℃下20分钟内完成。
与传统分子生物学检测方法相比，建立的检测方法操作更方便、更简单。该检测方法对每种圆环病毒的检测限为73.67拷贝/反应（通过概率回归分析，概率为95%），具有高特异性，与其他重要的猪病毒（包括PCV2）无交叉反应。组内和组间变异系数（CV）在2.08%至4.97%之间，表明具有高稳定性和可靠性。对60份临床样本和人工加标样本进行PCV3和PCV4定量聚合酶链反应（qPCR）的对比分析表明，一致性很高（kappa值分别为0.966和1，p<0.001），只有微小差异，验证了双重RAA检测方法在检测混合感染方面的有效性及其对PCV3和PCV4初步临床诊断的适用性。
本研究为使用RAA技术多重检测兽医病原体提供了有力依据，增强了该领域控制PCV3和PCV4的能力，并为新出现的圆环病毒的流行病学认识提供了可靠帮助。
